Generation of HPV-specific T-cells

ABSTRACT

Embodiments of the disclosure concern methods and compositions for immunotherapy for human papillomavirus infection and diseases associated therewith. In specific embodiments, methods concern production of immune cells that target one or more antigens of HPV16 and/or HPV18, including methods with stimulation steps that employ IL-7 and IL-15, but not IL-6 and/or IL-12. Other specific embodiments utilize stimulations in the presence of certain cells, such as costimulatory cells and certain antigen presenting cells.

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 62/395,440, filed Sep. 16, 2016, which is incorporated byreference herein in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under P50 CA097007 andPO1 CA94237 awarded by National Cancer Institute. The government hascertain rights in the invention.

TECHNICAL FIELD

The present disclosure concerns at least the fields of immunology, cellbiology, molecular biology, and medicine, including cancer medicine.

BACKGROUND

Human papillomavirus (HPV) is a DNA virus that establishes productiveinfections in keratinocytes of the skin or mucous membranes. There areover 170 types of HPV, a subset of which HPV types are carcinogenic,including high-risk sexually transmitted types that can develop intogenital neoplasias, including cervical intraepithelial neoplasia (CIN),vulvar intraepithelial neoplasia (VIN), penile intraepithelial neoplasia(PIN), and/or anal intraepithelial neoplasia (AIN), for example.HPV-induced cancers arise when viral sequences are integrated into thecellular DNA of host cells. Some of the HPV “early” genes, such as E6and E7, act as oncogenes that promote tumor growth and malignanttransformation.

Ramos et al., (J Immunother 2013; 36:66-76) describes a method forstimulating peripheral blood mononuclear cells to generate T-cellsspecific for HPV16 E6 and E7. In brief, the method comprises stimulationof peripheral blood mononuclear cells with dendritic cells in whichcells are cultured in CTL medium with or without IL-6, IL-7, IL-12 andIL-15, a second stimulation in which co-cultures are supplemented withIL-2, and weekly stimulation with pepmix-loaded accessory antigenpresenting cells (e.g., B-blasts) in the presence of IL-15.

The present disclosure provides relief for a long-felt need in the artto treat HPV-associated diseases, including at least for thoseassociated with HPV16 and HPV18, for example.

BRIEF SUMMARY

The present disclosure is directed to methods and compositions thatconcern immune system cells that are modified to immunogenicallyrecognize particular targets. In some embodiments, the presentdisclosure concerns the development of immune cells (including cytotoxicT-lymphocytes (CTLs, also referred to as cytotoxic T-cells)) that targeta biological moiety that elicits an immune response in an individual. Inspecific embodiments, the present disclosure concerns the development ofcytotoxic T-cells that target a HPV antigen, including a HPVdisease-associated antigen. In some cases, a mixture of cytotoxicT-cells is produced, and the mixture targets more than one HPV antigen,including more than one antigen of more than one HPV type, in somecases.

Embodiments of the disclosure concern methods and compositions forproviding therapy to individuals infected with HPV or that haveHPV-associated diseases, including cancers, for example. In specificembodiments, the disclosure regards methods and compositions foradoptive cellular immunotherapy that can target HPV-associated, e.g.,HPV16-associated and/or HPV18-associated, medical conditions (includingcancer) and are therapeutic therefor.

In certain aspects, the present disclosure concerns the development of aplurality of T-cells that target antigens from HPV, e.g., HPV16 and/orHPV18. The present disclosure provides significant and non-obviousimprovements on methods for generating T cell lines with specificityagainst HPV, e.g., HPV16 and/or HPV18 antigens.

In some embodiments of the disclosure, an individual is in need of themethods and/or compositions of the disclosure. In certain embodiments,an individual has been exposed to HPV, e.g., HPV16 and/or HPV18 (thepresence of which may or may not be known for the individual), or theindividual is suspected of having been exposed to or at risk for beingexposed to HPV, e.g., HPV16 and/or HPV18. In certain embodiments, theindividual has or is suspected of having or is at risk for havingHPV-associated disease, e.g., HPV16-associated and/or HPV18-associateddisease, including cancer.

In specific embodiments of part of the method, certain HPV, e.g., HPV16and/or HPV18, antigen(s) are presented to antigen-presenting cells(APCs) in the form of one or more peptides that span some or all ofcertain antigen(s). The antigenic peptides may be provided to theantigen-presenting cells in a library of peptide mixtures, which may bereferred to as pepmixes. In certain aspects of the disclosure, there ispooling of a variety of pepmixes for exposure to the APCs. APCs thatexpress the antigens may be exposed to peripheral blood T-cells undercertain conditions to result in stimulation of T-cells specific for thecertain HPV antigen(s).

Some aspects and embodiments of the present disclosure concern thegeneration and/or expansion of HPV-specific T-cells.

In a first aspect, the present disclosure provides a method forstimulating peripheral blood cells, preferably peripheral blood T-cells,wherein the method comprises stimulating peripheral blood T-cells withantigen presenting cells in the presence of interleukin (IL)-7 and IL-15and, in at least some cases, in the absence of IL-6 and/or IL-12,wherein the antigen presenting cells were previously exposed to one ormore peptides, wherein the peptides comprise sequence that correspondsto at least part of the sequence of one or more proteins of HPV.

In some embodiments a method of producing therapeutic T-cells for humanpapillomavirus (HPV)-associated disease(s) is provided, the methodcomprising the step of stimulating peripheral blood T-cells with antigenpresenting cells in the presence of one or more of interleukin IL-7 andIL-15 and, in at least some cases, in the absence of IL-6 and/or IL-12,wherein the antigen presenting cells were previously exposed to one ormore peptides, wherein the peptides comprise sequence that correspondsto at least part of the sequence of one or more proteins of HPV, whereinthe stimulating produces T-cells therapeutic for HPV-associateddiseases.

In some embodiments, the peripheral blood T-cells being stimulated areobtained from a prior stimulation of peripheral blood cells. The priorstimulation may comprise stimulating peripheral blood cells with antigenpresenting cells in the presence of IL-7 and IL-15, and in at least somecases in the presence of IL-6 and/or IL-12, wherein the antigenpresenting cells were previously exposed to one or more peptides,wherein the peptides comprise sequence that corresponds to at least partof the sequence of one or more proteins of HPV.

As such, prior to stimulating the peripheral blood T-cells, the methodsmay further comprise stimulating peripheral blood cells with antigenpresenting cells in the presence of IL-7 and IL-15, and in at least somecases in the presence of IL-6 and/or IL-12, wherein the antigenpresenting cells were previously exposed to one or more peptides,wherein the peptides comprise sequence that corresponds to at least partof the sequence of one or more proteins of HPV, to produce peripheralblood T-cells.

In some embodiments the one or more peptides comprise sequence thatcorresponds to at least part of the sequence of one or more proteins ofHPV16; one or more proteins of HPV18; or both of one or more proteins ofHPV16 and one or more proteins of HPV18. In some embodiments the one ormore peptides comprise sequence that corresponds to one or more ofproteins E5, E6, E7, L1 and L2. In some embodiments the one or morepeptides may be a library of peptides, including E1, E2, E3, E4, E5, E6,E7, L1, and/or L2 peptides.

In some embodiments the method may produce immune cells, such asT-cells, specific for HPV or for an HPV antigen. In some embodiments themethod may expand a population of T-cells present in the peripheralblood T-cells that are specific for HPV or for at least one HPV antigen.Immune cells other than T cells that may be produced by methods of thedisclosure including NK cells and NKT cells.

In some embodiments the antigen presenting cells are activated T-cells,dendritic cells (DC), B-Blasts (BB), or PBMCs, for example.

In some embodiments stimulation of peripheral blood T-cells in thepresence of IL-7 and IL-15 occurs in the absence of at least IL-2. Insome embodiments stimulation of peripheral blood T-cells in the presenceof IL-7 and IL-15 occurs in the absence of at least IL-4. In someembodiments stimulation of peripheral blood T-cells in the presence ofIL-7 and IL-15 occurs in the absence of at least IL-6. In someembodiments stimulation of peripheral blood T-cells in the presence ofIL-7 and IL-15 occurs in the absence of at least IL-12. In someembodiments stimulation of peripheral blood T-cells in the presence ofIL-7 and IL-15 occurs in the absence of at least IL-21. In someembodiments stimulation of peripheral blood T-cells in the presence ofIL-7 and IL-15 occurs in the absence of IL-6 and IL-12.

In some particular embodiments stimulation of cells in the method of thefirst aspect of the present invention occurs in the absence of IL-6 andIL-12.

In some embodiments, peripheral blood T-cells may be present in apopulation of peripheral blood mononuclear cells (PBMCs) or are obtainedor isolated therefrom. The PBMCs in the population may be non-adherentPBMCs. The antigen presenting cells may be activated T-cells, dendriticcells, B-blasts, or PBMCs, for example.

In a second aspect, the present disclosure provides a method forstimulating T-cells specific for HPV or for an HPV antigen, wherein themethod comprises stimulating T-cells specific for HPV or for an HPVantigen with antigen presenting cells in the presence of IL-7 and IL-15,and optionally in the presence of co-stimulatory cells, wherein theantigen presenting cells were previously exposed to one or morepeptides, wherein the peptides comprise sequence that corresponds to atleast part of the sequence of one or more proteins of HPV.

In some embodiments a method of producing therapeutic T-cells for humanpapillomavirus (HPV)-associated diseases is provided, the methodcomprising the step of stimulating T-cells specific for HPV or for anHPV antigen with antigen presenting cells in the presence of one or moreof interleukin IL-7 and IL-15, and optionally in the presence ofco-stimulatory cells, wherein the antigen presenting cells werepreviously exposed to one or more peptides, wherein the peptidescomprise sequence that corresponds to at least part of the sequence ofone or more proteins of HPV, wherein the stimulating produces T-cellstherapeutic for one or more HPV-associated diseases.

In some embodiments the antigen presenting cells are activated T-cells,dendritic cells (DC), B-Blasts (BB) or PBMCs. In particular embodimentsthe antigen presenting cells are activated T-cells.

In some embodiments the co-stimulatory cells are one or more cell typesselected from the group consisting of CD80+ cells, CD86+ cells, CD83+cells, 4-1BBL+ cells, CD40+ cells, OX40+ cells, and a combinationthereof. The co-stimulatory cells may be CD80+/CD86+/CD83+/4-1BBL+cells.

In some embodiments the stimulation of T-cells specific for HPV or foran HPV antigen is not a first stimulation step. The T-cells beingstimulated cells may be the product of a prior stimulation, e.g. usingthe method of the first aspect of the present disclosure.

In some embodiments the one or more peptides comprise sequence thatcorresponds to at least part of the sequence of one or more proteins ofHPV16; one or more proteins of HPV18; or one or more proteins of HPV16and one or more proteins of HPV18. In some embodiments the one or morepeptides comprise sequence that corresponds to one or more of proteinsE5, E6, E7, L1 and L2. In some embodiments the one or more peptides maybe a library of peptides, including E1, E2, E3, E4, E5, E6, E7, L1,and/or L2 peptides.

In some embodiments the method may produce T-cells specific for HPV orfor an HPV antigen. In some embodiments the method may expand apopulation of T-cells specific for HPV or for an HPV antigen.

In certain embodiments stimulation of T-cells specific for HPV or for anHPV antigen comprises stimulating T-cells specific for HPV or for an HPVantigen with antigen presenting cells in the presence of IL-7, IL-15,and in the presence of one or more types of co-stimulatory cells.

In some embodiments stimulation of T-cells in the presence of IL-7 andIL-15 is in the absence of IL-2. In some embodiments stimulation ofT-cells in the presence of IL-7 and IL-15 is in the absence of IL-4. Insome embodiments stimulation of T-cells in the presence of IL-7 andIL-15 is in the absence of IL-6. In some embodiments stimulation ofT-cells in the presence of IL-7 and IL-15 is in the absence of IL-7. Insome embodiments stimulation of T-cells in the presence of IL-7 andIL-15 is in the absence of IL-12. In some embodiments stimulation ofT-cells in the presence of IL-7 and IL-15 is in the absence of IL-21. Insome embodiments stimulation of T-cells in the method of the firstaspect of the present invention is in the absence of IL-6 and IL-12.

Methods according to the first and second aspect of the presentdisclosure may be methods of producing therapeutic T-cells forHPV-associated diseases. The stimulation of cells may produce T-cellsthat are therapeutic for HPV-associated diseases.

In a third aspect, the methods of the first and second aspects may becombined to provide a method of producing therapeutic T-cells forHPV-associated diseases, the method comprising:

stimulating peripheral blood cells, preferably peripheral blood T-cells,wherein the method comprises stimulating peripheral blood T-cells withantigen presenting cells in the presence of interleukin (IL)-7 andIL-15, and optionally in the absence of IL-6 and/or IL-12, wherein theantigen presenting cells were previously exposed to one or morepeptides, wherein the peptides comprise sequence that corresponds to atleast part of the sequence of one or more proteins of HPV;

stimulating T-cells obtained from (i) with antigen presenting cells inthe presence of interleukin (IL)-7 and IL-15, and optionally in thepresence of one or more types of co-stimulatory cells, wherein theantigen presenting cells were previously exposed to one or morepeptides, wherein the peptides comprise sequence that corresponds to atleast part of the sequence of one or more proteins of HPV.

In some embodiments prior to step (ii), T-cells obtained from (i) may bere-stimulated in the presence of IL-7 and IL-15 but not in the presenceof co-stimulatory cells, and optionally in the absence of IL-6 and/orIL-12. Such re-stimulation may occur for one, two, three, four, five ormore rounds, as required.

In some embodiments the antigen presenting cells used in (i) aredendritic cells (DC), B-Blasts (BB) or PBMCs. In some embodiments theantigen presenting cells used in (ii) are activated T-cells, dendriticcells (DC), B-Blasts (BB) or PBMCs. In some embodiments the antigenpresenting cells used in (i) are different to the antigen presentingcells used in (ii), although they may be the same in certain cases. Inparticular embodiments the antigen presenting cells used in (ii) areactivated T-cells.

In some embodiments the co-stimulatory cells are one or more cell typesselected from the group consisting of CD80+ cells, CD86+ cells, CD83+cells, 4-1BBL+ cells, CD40+ cells, OX40+ cells, and a combinationthereof. The co-stimulatory cells may be CD80+/CD86+/CD83+/4-1BBL+cells.

In some embodiments stimulation of cells in the presence of IL-7 andIL-15 is in the absence of IL-2. In some embodiments stimulation ofcells in the presence of IL-7 and IL-15 is in the absence of IL-4. Insome embodiments stimulation of cells in the presence of IL-7 and IL-15is in the absence of IL-6. In some embodiments stimulation of cells inthe presence of IL-7 and IL-15 is in the absence of IL-12. In someembodiments stimulation of cells in the presence of IL-7 and IL-15 is inthe absence of IL-21. In some embodiments stimulation of cells in thepresence of IL-7 and IL-15 is in the absence of IL-6 and IL-12.

In some preferred embodiments stimulation of cells in step (i) is in theabsence of IL-6 and IL-12.

In some embodiments stimulation of cells in step (ii) is in the absenceof IL-6 and IL-12.

Accordingly, in some embodiments a method of producing therapeuticT-cells for HPV-associated diseases is provided, the method comprising:

(i) stimulating peripheral blood cells, wherein the method comprisesstimulating peripheral blood T-cells with antigen presenting cells inthe presence of interleukin (IL)-7 and IL-15 and optionally in theabsence of IL-6 and/or IL-12, wherein the antigen presenting cells werepreviously exposed to one or more peptides, wherein the peptidescomprise sequence that corresponds to at least part of the sequence ofone or more proteins of HPV;

(ii) stimulating T-cells obtained from (i) with antigen presenting cellsin the presence of interleukin (IL)-7 and IL-15 and optionally in theabsence of IL-6 and/or IL-12, wherein the antigen presenting cells werepreviously exposed to one or more peptides, wherein the peptidescomprise sequence that corresponds to at least part of the sequence ofone or more proteins of HPV, wherein (ii) is optionally repeated one ormore times; and

(iii) stimulating T-cells obtained from (ii) with antigen presentingcells in the presence of IL-7 and IL-15, and optionally in the presenceof co-stimulatory cells, wherein the antigen presenting cells werepreviously exposed to one or more peptides, wherein the peptidescomprise sequence that corresponds to at least part of the sequence ofone or more proteins of HPV, wherein (iii) is optionally repeated one ormore times.

In some embodiments the antigen presenting cells used in (i) and (ii)are dendritic cells (DC) B-Blasts (BB) or PBMCs. In some embodiments theantigen presenting cells used in (iii) are activated T-cells, dendriticcells (DC), B-Blasts (BB) or PBMCs. In some embodiments the antigenpresenting cells used in (iii) are different to the antigen presentingcells used in (i) and/or (ii). In preferred embodiments the antigenpresenting cells used in (iii) are activated T-cells.

In preferred embodiments the stimulation in (iii) is in the presence ofco-stimulatory cells. In some embodiments the co-stimulatory cells areone or more cell types selected from the group consisting of CD80+cells, CD86+ cells, CD83+ cells, 4-1BBL+ cells, CD40+ cells, OX40+cells, and a combination thereof. The co-stimulatory cells may beCD80+/CD86+/CD83+/4-1BBL+ cells.

In some embodiments stimulation of cells in the presence of IL-7 andIL-15 is in the absence of IL-2. In some embodiments stimulation ofcells in the presence of IL-7 and IL-15 is in the absence of IL-4. Insome embodiments stimulation of cells in the presence of IL-7 and IL-15is in the absence of IL-6. In some embodiments stimulation of cells inthe presence of IL-7 and IL-15 is in the absence of IL-12. In someembodiments stimulation of cells in the presence of IL-7 and IL-15 is inthe absence of IL-21. In some embodiments stimulation of cells in thepresence of IL-7 and IL-15 is in the absence of IL-6 and IL-12.

In some embodiments stimulation of cells in step (i) is in the absenceof IL-6 and IL-12. In some other embodiments stimulation of cells instep (i) is in the presence of IL-6 and IL-12.

In some particular embodiments stimulation of cells in step (ii) is inthe absence of IL-6 and IL-12. In some particular embodimentsstimulation of cells in step (iii) is in the absence of IL-6 and IL-12.

In some particular embodiments methods of the present disclosure are forproducing T-cells specific for HPV16 and/or HPV18. In some particularembodiments methods of the present invention are for producing T-cellsspecific for HPV16-associated and/or HPV18-associated diseases.

In some embodiments, peripheral blood T-cells may be obtained from anindividual that is known to be infected or suspected of being infectedwith HPV; HPV16 or HPV18; or both HPV16 and HPV18.

In some embodiments, antigen presenting cells may be obtained from anindividual that is known to be infected or suspected of being infectedwith HPV; HPV16 or HPV18; or both HPV16 and HPV18.

In some embodiments, the method may occur in the absence of exposing theT-cells produced by the method to activated B cells that were previouslyexposed to a library of peptides.

In some embodiments, antigen presenting cells may be autologous orallogeneic to an individual intended to be treated with the therapeuticT-cells obtained.

In some embodiments, the one or more peptides comprise sequence thatcorresponds to at least part of the sequence of one or more proteins ofHPV16; one or more proteins of HPV18; or one or more proteins of HPV16and one or more proteins of HPV18. In some embodiments the one or morepeptides comprise sequence that corresponds to one or more of proteinsE1, E2, E3, E4, E5, E6, E7, L1, and/or L2 that come from HPV16, HPV18,or HPV16 and HPV18.

In embodiments of the present disclosure the peptides may comprisesequence that corresponds to one or more of HPV proteins E1, E2, E3, E4,E5, E6, E7, L1, and/or L2. The peptides may correspond to a contiguousamino acid sequence present within said HPV protein. A peptide may havea length of at least or no more than 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, or 20 amino acids in length, or of 15 amino acids in length.The collection of peptides may form a library and peptides in thelibrary may overlap in sequence with other peptides by any suitableamount, including 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 aminoacids, for example. The peptides may comprise sequence that correspondsto: a) the HPV18 E6 protein and/or the HPV18 E7 protein, and/or b) theHPV16 E6 protein and/or the HPV16 E7 protein.

In embodiments of the present disclosure the HPV may be HPV16 or HPV18,or both. In embodiments concerned with treatment of an HPV-associateddisease, the disease may be cancer and the peptides may comprise asequence that corresponds to one or both of E6 and E7. When theHPV-associated disease is a pre-cancerous lesion, the peptides maycomprise sequence that corresponds to one, some, or all of E1, E2, E3,E4, E5, E6, E7, L1, and L2.

T-cells produced by the methods of the present disclosure may beisolated and/or purified, e.g., isolated/purified from other cells.

In some embodiments, a therapeutically effective amount of T-cellsproduced by the methods of the present disclosure are provided to anindividual that has been exposed to HPV, or that has HPV-associateddisease. In a related aspect T-cells produced by the method of thepresent disclosure are provided for use in the treatment ofHPV-associated disease. In another related aspect the use of T-cellsproduced by the method of the present disclosure are provided for use inthe manufacture of a medicament for use in the treatment ofHPV-associated disease.

In one aspect of the present invention T-cells for use in a method ofadoptive cellular immunotherapy are provided, wherein the T-cells areobtained by, obtainable by, or are the product of, a method forstimulating peripheral blood or T-cells or a method of producingtherapeutic T-cells described herein, the method of adoptive cellularimmunotherapy comprising administering the T-cells to the subject.

In one aspect of the present invention the use of T-cells in themanufacture of a medicament for use in a method of adoptive cellularimmunotherapy comprising administering the T-cells to the subject isprovided, wherein the T-cells are obtained by, obtainable by, or are theproduct of, a method for stimulating peripheral blood or T-cells or amethod of producing therapeutic T-cells described herein.

In one aspect of the present invention a method of preparing apharmaceutical composition, medicament or vaccine is provided, themethod comprising stimulating peripheral blood or T-cells according to amethod described herein, or producing therapeutic T-cells according to amethod described herein, and mixing the cells obtained, with apharmaceutically acceptable carrier, adjuvant, diluent or excipient.

The disease to be treated may be a neoplasm. The neoplasm may be acancer. The cancer may be an HPV-positive cancer, HPV16-positive cancerand/or HPV18-positive cancer.

The individual to be treated may be a human. The individual may be apatient. The individual may have been exposed to HPV, such as HPV16,HPV18, or both HPV16 and HPV18, or has an HPV-, HPV16- and/orHPV18-associated disease. The HPV-, HPV16- and/or HPV18-associateddisease may be a neoplasm. The neoplasm may be a cancer.

A cancer may be of any kind. In some embodiments the cancer is acervical cancer, anal cancer, vulvar cancer, vaginal cancer, penilecancer, or oropharyngeal cancer. In some embodiments the cancer is aHPV-related carcinoma, HPV-positive oropharyngeal carcinoma,HPV-positive cervical carcinoma, HPV-positive anal carcinoma,HPV-positive vulvar carcinoma, nasopharyngeal carcinoma, HPV-positivepenile carcinoma, HPV-positive dysplasias of any site, or laryngealpapillomatosis.

The individual or subject may have received, be receiving, or willreceive an additional cancer therapy. The additional cancer therapy maybe surgery, radiation, hormone therapy, chemotherapy, immunotherapy, ora combination thereof.

The individual or subject may be determined as having HPV-associatedcancer or HPV-positive cancer. The individual may be determined ashaving HPV16-associated cancer or HPV16-positive cancer. The individualmay be determined as having HPV18-associated cancer or HPV18-positivecancer. The individual or subject may be any animal or human. Theindividual or subject is preferably mammalian, more preferably human.The individual or subject may be a non-human mammal, but is morepreferably human. The individual or subject may be male or female. Theindividual or subject may be a patient.

Methods according to the present disclosure that involve steps of cellstimulation may be performed in vitro or ex vivo. The term “in vitro” isintended to encompass studies with materials, biological substances,cells and/or tissues in laboratory conditions or in culture. “Ex vivo”refers to something present or taking place outside an organism, e.g.outside the human or animal body, which may be on tissue (e.g. wholeorgans) or cells taken from the organism.

In one embodiment, there is a method for stimulating peripheral bloodcells, the method comprising stimulating peripheral blood T-cells withantigen presenting cells in the presence of interleukin (IL)-7 and IL-15and in the absence of IL-6 and/or IL-12, wherein the antigen presentingcells were previously exposed to one or more peptides, wherein thepeptides comprise sequence that corresponds to at least part of thesequence of one or more proteins of human papillomavirus (HPV). Theperipheral blood T-cells may be obtained from a prior stimulation ofperipheral blood cells, such as wherein the prior stimulation ofperipheral blood cells comprises stimulating peripheral blood cells withantigen presenting cells in the presence of IL-7 and IL-15, and in thepresence of IL-6 and/or IL-12, wherein the antigen presenting cells werepreviously exposed to one or more peptides, wherein the peptidescomprise sequence that corresponds to at least part of the sequence ofone or more proteins of HPV. In specific cases, prior to stimulating theperipheral blood T-cells, the method further comprises stimulatingperipheral blood cells with antigen presenting cells in the presence ofIL-7 and IL-15, and in the presence of IL-6 and/or IL-12, wherein theantigen presenting cells were previously exposed to one or morepeptides, wherein the peptides comprise sequence that corresponds to atleast part of the sequence of one or more proteins of HPV, to produceperipheral blood T-cells.

In an embodiment, there is a method of producing therapeutic T-cells forHPV-associated diseases, the method comprising the step of: stimulatingperipheral blood T-cells with antigen presenting cells in the presenceof one or more of IL-7 and IL-15 and in the absence of IL-6 and/orIL-12, wherein the antigen presenting cells were previously exposed toone or more peptides, wherein the peptides comprise sequence thatcorresponds to at least part of the sequence of one or more proteins ofHPV, wherein the stimulating produces T-cells therapeutic forHPV-associated diseases. The peripheral blood T-cells may be obtainedfrom a prior stimulation of peripheral blood cells, such as wherein theprior stimulation of peripheral blood cells comprises stimulatingperipheral blood cells with antigen presenting cells in the presence ofIL-7 and IL-15, and in the presence of IL-6 and/or IL-12, wherein theantigen presenting cells were previously exposed to one or morepeptides, wherein the peptides comprise sequence that corresponds to atleast part of the sequence of one or more proteins of HPV. In specificcases, prior to stimulating the peripheral blood T-cells, the methodfurther comprises stimulating peripheral blood cells with antigenpresenting cells in the presence of IL-7 and IL-15, and in the presenceof IL-6 and/or IL-12, wherein the antigen presenting cells werepreviously exposed to one or more peptides, wherein the peptidescomprise sequence that corresponds to at least part of the sequence ofone or more proteins of HPV, to produce peripheral blood T-cells.

In embodiments of methods encompassed by the disclosure, antigenpresenting cells are activated T-cells, dendritic cells, B-blasts, orPBMCs. Peripheral blood T-cells may be present in a population ofperipheral blood mononuclear cells (PBMCs) or are obtained or isolatedtherefrom, in at least some cases, and the PBMCs in the population maybe non-adherent PBMCs. When employed, co-stimulatory cells may be CD80+,CD86+, CD83+, 4-1BBL+, CD40+ cells, OX40+ cells, or a combinationthereof.

In a particular embodiment, there is a method for stimulating T-cellsspecific for HPV or for an HPV antigen, the method comprisingstimulating T-cells specific for HPV or for an HPV antigen with antigenpresenting cells in the presence of IL-7 and IL-15 and in the presenceof co-stimulatory cells, wherein the antigen presenting cells werepreviously exposed to one or more peptides, wherein the peptidescomprise sequence that corresponds to at least part of the sequence ofone or more proteins of HPV.

In certain embodiments, there is a method of producing therapeuticT-cells for HPV-associated diseases, the method comprising the step ofstimulating T-cells specific for HPV or for an HPV antigen with antigenpresenting cells in the presence of one or more of IL-7 and IL-15 and inthe presence of co-stimulatory cells, wherein the antigen presentingcells were previously exposed to one or more peptides, wherein thepeptides comprise sequence that corresponds to at least part of thesequence of one or more proteins of HPV, wherein the stimulatingproduces T-cells therapeutic for HPV-associated diseases.

In one embodiment, there is a method of producing therapeutic T-cellsfor HPV-associated diseases, the method comprising: (i) stimulatingperipheral blood cells, wherein the method comprises stimulatingperipheral blood T-cells with antigen presenting cells in the presenceof IL-7 and IL-15 and optionally in the absence of IL-6 and/or IL-12,wherein the antigen presenting cells were previously exposed to one ormore peptides, wherein the peptides comprise sequence that correspondsto at least part of the sequence of one or more proteins of HPV; and(ii) stimulating T-cells obtained from (i) with antigen presenting cellsin the presence of IL-7 and IL-15, and in the presence of co-stimulatorycells, wherein the antigen presenting cells were previously exposed toone or more peptides, wherein the peptides comprise sequence thatcorresponds to at least part of the sequence of one or more proteins ofHPV. In specific embodiments, prior to step (ii) T-cells obtained from(i) may be re-stimulated in the presence of IL-7 and IL-15 but not inthe presence of co-stimulatory cells.

In an embodiment, a method of producing therapeutic T-cells forHPV-associated diseases is provided, the method comprising: (i)stimulating peripheral blood cells, wherein the method comprisesstimulating peripheral blood T-cells with antigen presenting cells inthe presence of interleukin (IL)-7 and IL-15 and optionally in theabsence of IL-6 and/or IL-12, wherein the antigen presenting cells werepreviously exposed to one or more peptides, wherein the peptidescomprise sequence that corresponds to at least part of the sequence ofone or more proteins of HPV; (ii) stimulating T-cells obtained from (i)with antigen presenting cells in the presence of interleukin (IL)-7 andIL-15 and optionally in the absence of IL-6 and/or IL-12, wherein theantigen presenting cells were previously exposed to one or morepeptides, wherein the peptides comprise sequence that corresponds to atleast part of the sequence of one or more proteins of HPV, wherein (ii)is optionally repeated one or more times; and (iii) stimulating T-cellsobtained from (ii) with antigen presenting cells in the presence ofinterleukin (IL)-7 and IL-15, and in the presence of co-stimulatorycells, wherein the antigen presenting cells were previously exposed toone or more peptides, wherein the peptides comprise sequence thatcorresponds to at least part of the sequence of one or more proteins ofHPV, wherein (iii) is optionally repeated one or more times.

In any method of the disclosure, the HPV may be HPV16 or HPV18. Peptidescomprising sequence that corresponds to one or more of E1, E2, E3, E4,E5, E6, E7, L1, and L2 may be utilized in any method of the disclosure.The peptides may comprise sequence that corresponds to: a) the HPV18 E6protein and/or the HPV18 E7 protein, and/or b) the HPV16 E6 proteinand/or the HPV16 E7 protein. In some cases, an individual being providedwith an effective amount of cells as described herein has anHPV-associated disease, such as cancer, and the peptides comprisesequence that corresponds to one or both of E6 and E7. In specificaspects, the one or more peptides comprises peptides of at least or nomore than 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 aminoacids in length, and in particular the one or more peptides comprisespeptides of 15 amino acids in length. In specific embodiments, one ormore peptides form a library and peptides in the library overlap insequence with other peptides by 11 amino acids.

In particular embodiments, a therapeutically effective amount of T-cellsproduced by the method are provided to an individual that has beenexposed to HPV or that has HPV-associated disease. In specificembodiments, an HPV-associated disease comprises a neoplasm.

A therapeutically effective amount of T-cells produced by a method ofthe disclosure may be provided to an individual that has been exposed toHPV16, HPV18, or both, or that has HPV16-associated and/orHPV18-associated disease, including a neoplasm such as cancer.

In particular embodiments, the cancer is a cervical cancer, anal cancer,vulvar cancer, vaginal cancer, penile cancer, oropharyngeal cancer,nasopharyngeal carcinoma, laryngeal papillomatosis, laryngeal cancer,head and neck cancer, or a dysplasia of any site thereof.

In some cases, an individual that has received and/or will receive cellsof the disclosure has also received, is receiving, or will receive anadditional cancer therapy, such as surgery, radiation, hormone therapy,chemotherapy, immunotherapy, or a combination thereof.

In certain aspects, an individual that has received and/or will receivecells of the disclosure is determined as having HPV-associated cancer,such as HPV16-associated cancer or HPV18-associated cancer.

The following numbered paragraphs contain statements of broadcombinations of the inventive technical features herein disclosed:

1. A method for stimulating peripheral blood cells, the methodcomprising stimulating peripheral blood T-cells with antigen presentingcells in the presence of interleukin (IL)-7 and IL-15 and in the absenceof IL-6 and/or IL-12, wherein the antigen presenting cells werepreviously exposed to one or more peptides, wherein the peptidescomprise sequence that corresponds to at least part of the sequence ofone or more proteins of human papillomavirus (HPV).

2. A method of producing therapeutic T-cells for HPV-associateddiseases, the method comprising the step of:

-   -   stimulating peripheral blood T-cells with antigen presenting        cells in the presence of one or more of IL-7 and IL-15 and in        the absence of IL-6 and/or IL-12, wherein the antigen presenting        cells were previously exposed to one or more peptides, wherein        the peptides comprise sequence that corresponds to at least part        of the sequence of one or more proteins of HPV,    -   wherein the stimulating produces T-cells therapeutic for        HPV-associated diseases.

3. The method of paragraph 1 or 2, wherein the peripheral blood T-cellsare obtained from a prior stimulation of peripheral blood cells.

4. The method of paragraph 3, wherein the prior stimulation ofperipheral blood cells comprises stimulating peripheral blood cells withantigen presenting cells in the presence of IL-7 and IL-15, and in thepresence of IL-6 and/or IL-12, wherein the antigen presenting cells werepreviously exposed to one or more peptides, wherein the peptidescomprise sequence that corresponds to at least part of the sequence ofone or more proteins of HPV.

5. The method of paragraph 1 or 2, wherein prior to stimulating saidperipheral blood T-cells, the method further comprises stimulatingperipheral blood cells with antigen presenting cells in the presence ofIL-7 and IL-15, and in the presence of IL-6 and/or IL-12, wherein theantigen presenting cells were previously exposed to one or morepeptides, wherein the peptides comprise sequence that corresponds to atleast part of the sequence of one or more proteins of HPV, to produceperipheral blood T-cells.

6. The method of any one of paragraphs 1-7, wherein the antigenpresenting cells are dendritic cells, B-blasts, or PBMCs.

7. The method of any one of paragraphs 1-6, wherein the peripheral bloodT-cells are present in a population of peripheral blood mononuclearcells (PBMCs) or are obtained or isolated therefrom.

8. The method of paragraph 7, wherein the PBMCs in the population arenon-adherent PBMCs.

9. A method for stimulating T-cells specific for HPV or for an HPVantigen, the method comprising stimulating T-cells specific for HPV orfor an HPV antigen with antigen presenting cells in the presence of IL-7and IL-15 and in the presence of co-stimulatory cells, wherein theantigen presenting cells were previously exposed to one or morepeptides, wherein the peptides comprise sequence that corresponds to atleast part of the sequence of one or more proteins of HPV.

10. A method of producing therapeutic T-cells for HPV-associateddiseases, the method comprising the step of stimulating T-cells specificfor HPV or for an HPV antigen with antigen presenting cells in thepresence of one or more of IL-7 and IL-15 and in the presence ofco-stimulatory cells, wherein the antigen presenting cells werepreviously exposed to one or more peptides, wherein the peptidescomprise sequence that corresponds to at least part of the sequence ofone or more proteins of HPV, wherein the stimulating produces T-cellstherapeutic for HPV-associated diseases.

11. The method of paragraph 9 or 10, wherein the antigen presentingcells are activated T cells, dendritic cells, B-blasts, or PBMCs.

12. The method of any one of paragraphs 9 to 11, wherein theco-stimulatory cells are CD80+, CD86+, CD83+, 4-1BBL+, CD40+ cells,OX40+ cells, or a combination thereof.

13. A method of producing therapeutic T-cells for HPV-associateddiseases, the method comprising:

-   -   (i) stimulating peripheral blood cells, wherein the method        comprises stimulating peripheral blood T-cells with antigen        presenting cells in the presence of IL-7 and IL-15 and        optionally in the absence of IL-6 and/or IL-12, wherein the        antigen presenting cells were previously exposed to one or more        peptides, wherein the peptides comprise sequence that        corresponds to at least part of the sequence of one or more        proteins of HPV; and    -   (ii) stimulating T-cells obtained from (i) with antigen        presenting cells in the presence of IL-7 and IL-15, and in the        presence of co-stimulatory cells, wherein the antigen presenting        cells were previously exposed to one or more peptides, wherein        the peptides comprise sequence that corresponds to at least part        of the sequence of one or more proteins of HPV.

14. The method of paragraph 13, wherein prior to step (ii) T-cellsobtained from (i) may be re-stimulated in the presence of IL-7 and IL-15but not in the presence of co-stimulatory cells.

15. The method of paragraph 13 or 14, wherein the antigen presentingcells used in (i) are dendritic cells (DC), B-Blasts (BB) or PBMCs.

16. The method of any one of paragraphs 13 to 15, wherein the antigenpresenting cells used in (ii) are activated T cells, dendritic cells(DC) or B-Blasts (BB).

17. The method of any one of paragraphs 13 to 16, wherein theco-stimulatory cells are CD80+, CD86+, CD83+, 4-1BBL+, CD40+ cells,OX40+ cells, or a combination thereof.

18. A method of producing therapeutic T-cells for HPV-associateddiseases is provided, the method comprising:

-   -   (i) stimulating peripheral blood cells, wherein the method        comprises stimulating peripheral blood T-cells with antigen        presenting cells in the presence of interleukin (IL)-7 and IL-15        and optionally in the absence of IL-6 and/or IL-12, wherein the        antigen presenting cells were previously exposed to one or more        peptides, wherein the peptides comprise sequence that        corresponds to at least part of the sequence of one or more        proteins of HPV;    -   (ii) stimulating T-cells obtained from (i) with antigen        presenting cells in the presence of interleukin (IL)-7 and IL-15        and optionally in the absence of IL-6 and/or IL-12, wherein the        antigen presenting cells were previously exposed to one or more        peptides, wherein the peptides comprise sequence that        corresponds to at least part of the sequence of one or more        proteins of HPV, wherein (ii) is optionally repeated one or more        times; and    -   (iii) stimulating T-cells obtained from (ii) with antigen        presenting cells in the presence of interleukin (IL)-7 and        IL-15, and in the presence of co-stimulatory cells, wherein the        antigen presenting cells were previously exposed to one or more        peptides, wherein the peptides comprise sequence that        corresponds to at least part of the sequence of one or more        proteins of HPV, wherein (iii) is optionally repeated one or        more times.

19. The method of paragraph 18, wherein the antigen presenting cellsused in (i) and (ii) are DC, BB, or PBMCs.

20. The method of paragraph 18 or 19, wherein the antigen presentingcells used in (iii) are activated T cells, DC, BB, or PBMCs.

21. The method of any one of paragraphs 18 to 20, wherein theco-stimulatory cells are CD80+, CD86+, CD83+, 4-1BBL+, CD40+ cells,OX40+ cells or a combination thereof.

22. The method of any one of paragraphs 1-21, wherein the HPV is HPV16or HPV18.

23. The method of any one of paragraphs 1-22, wherein the peptidescomprise sequence that corresponds to one or more of E1, E2, E3, E4, E5,E6, E7, L1, and L2.

24. The method of any one of paragraphs 1-23, wherein the HPV-associateddisease is cancer and the peptides comprise sequence that corresponds toone or both of E6 and E7.

25. The method of any one of paragraphs 1-25, wherein the peptidescomprise sequence that corresponds to:

-   -   a) the HPV18 E6 protein and/or the HPV18 E7 protein, and/or    -   b) the HPV16 E6 protein and/or the HPV16 E7 protein.

26. The method of any one of paragraphs 1-25, wherein the one or morepeptides comprises peptides of at least or no more than 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length.

27. The method of any one of paragraphs 1-26, wherein the one or morepeptides comprises peptides of 15 amino acids in length.

28. The method of any one of paragraphs 1-27, wherein one or morepeptides form a library and peptides in the library overlap in sequencewith other peptides by 11 amino acids.

29. The method of any one of paragraphs 1-28, wherein a therapeuticallyeffective amount of T-cells produced by the method are provided to anindividual that has been exposed to HPV or that has HPV-associateddisease.

30. The method of paragraph 29, wherein the HPV-associated diseasecomprises a neoplasm.

31. The method of any one of paragraphs 1 to 30, wherein atherapeutically effective amount of T-cells produced by the method areprovided to an individual that has been exposed to HPV16, HPV18 or both,or that has HPV16-associated and/or HPV18-associated disease.

32. The method of paragraph 31, wherein the HPV16-associated and/orHPV18-associated disease comprises a neoplasm.

33. The method of paragraph 31 or 32, wherein the neoplasm is cancer.

34. The method of paragraph 33, wherein the cancer is cervical cancer,anal cancer, vulvar cancer, vaginal cancer, penile cancer, oropharyngealcancer, nasopharyngeal carcinoma, laryngeal papillomatosis, laryngealcancer, head and neck cancer, or a dysplasia of any of site thereof.

35. The method of paragraph 33 or 34, wherein the individual hasreceived, is receiving, or will receive an additional cancer therapy.

36. The method of paragraph 35, wherein the additional cancer therapy issurgery, radiation, hormone therapy, chemotherapy, immunotherapy, or acombination thereof.

37. The method of any one of paragraphs 33 to 36, wherein the individualis determined as having HPV-associated cancer.

38. The method of any one of paragraphs 33 to 37, wherein the individualis determined as having HPV16-associated cancer.

39. The method of any one of paragraphs 33 to 38, wherein the individualis determined as having HPV18-associated cancer.

40. T-cells for use in a method of adoptive cellular immunotherapy,wherein the T-cells are obtained by, obtainable by, or are the productof, a method for stimulating peripheral blood or T-cells or a method ofproducing therapeutic T-cells according to any one of paragraphs 1 to39, wherein the method of adoptive cellular immunotherapy comprisesadministering the T-cells to the subject.

41. Use of T-cells in the manufacture of a medicament for use in amethod of adoptive cellular immunotherapy comprising administering theT-cells to the subject, wherein the T-cells are obtained by, obtainableby, or are the product of, a method for stimulating peripheral blood orT-cells or a method of producing therapeutic T-cells according to anyone of claims 1 to 39.

42. A method of preparing a pharmaceutical composition, medicament orvaccine, the method comprising stimulating peripheral blood or T-cellsor producing therapeutic T-cells according to any one of claims 1 to 39,and mixing the cells obtained with a pharmaceutically acceptablecarrier, adjuvant, diluent or excipient.

The foregoing has outlined rather broadly the features and technicaladvantages of the present invention in order that the detaileddescription of the invention that follows may be better understood.Additional features and advantages of the invention will be describedhereinafter which form the subject of the claims of the invention. Itshould be appreciated by those skilled in the art that the conceptionand specific embodiment disclosed may be readily utilized as a basis formodifying or designing other structures for carrying out the samepurposes of the present invention. It should also be realized by thoseskilled in the art that such equivalent constructions do not depart fromthe spirit and scope of the invention as set forth in the appendedclaims. The novel features which are believed to be characteristic ofthe invention, both as to its organization and method of operation,together with further objects and advantages will be better understoodfrom the following description when considered in connection with theaccompanying figures. It is to be expressly understood, however, thateach of the figures is provided for the purpose of illustration anddescription only and is not intended as a definition of the limits ofthe present invention.

The invention includes the combination of the aspects and preferredfeatures described except where such a combination is clearlyimpermissible or expressly avoided.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

Aspects and embodiments of the present invention will now beillustrated, by way of example, with reference to the accompanyingfigures. Further aspects and embodiments will be apparent to thoseskilled in the art. All documents mentioned in this text areincorporated herein by reference.

Throughout this specification, including the claims which follow, unlessthe context requires otherwise, the word “comprise,” and variations suchas “comprises” and “comprising,” will be understood to imply theinclusion of a stated integer or step or group of integers or steps butnot the exclusion of any other integer or step or group of integers orsteps.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A demonstrates a method in the art that utilizes certainconditions for the production of HPV16-specific T-cells. FIG. 1A is abar chart showing production of spot forming colonies (SFC) onstimulation of PBMCs from three HPV-associated cancer patients(identified as OCV, HND and HNC) with autologous DCs loaded with nopepmix (Neg), HPV16 E6 pepmix (HPV16 E6) or HPV16 E7 pepmix (HPV16 E7).For patient OCV the three bars present from left to right are Neg, HPV16E6 and HPV16 E7. For patients HND and HNC the two bars present from leftto right are HPV16 E6 and HPV16 E7.

FIG. 1B demonstrates a method of the disclosure that utilizes, undercertain novel conditions, the production of a mixture of T-cellsspecific for HPV16 or HPV18 by stimulation of T-cells in the presence ofIL-7 and IL-15 and in the absence of IL-6 and IL-12. FIG. 1B is a barchart showing production of spot forming colonies (SFC) on stimulationof PBMCs from three HPV-associated cancer patients (identified as OCV,PCV and HND). For patient OCV the five bars present from left to rightare no pepmix (Neg), HPV16 E6 pepmix (HPV16 E6), HPV16 E7 pepmix (HPV16E7), HPV18 E6 pepmix (HPV18 E6), and HPV18 E7 pepmix (HPV18 E7). Forpatient PCD the two bars present from left to right are HPV16 E6 andHPV16 E7. For patient HND the four bars present from left to right areHPV16 E6, HPV16 E7, HPV18 E6 and HPV18 E7.

FIG. 2 is a chart showing in vivo expansion and persistence of infusedHPV stimulated T-cells transduced with a dominant negative receptor forTGF-beta (DNRII) in patient #1 at time points post infusion.

FIG. 3 is a chart showing in vivo expansion and persistence of infusedHPV stimulated T cells transduced with a dominant negative receptor forTGF-beta (DNRII) in patient #2 at time points post infusion.

FIG. 4 shows PET scans (left and center) and photographs of physicalexamination (right) for patient #2. Top row: pre-treatment. Bottom row:6 weeks after treatment with HPV stimulated T cells produced accordingto the present invention.

DETAILED DESCRIPTION

The scope of the present application is not intended to be limited tothe particular embodiments of the process, machine, manufacture,composition of matter, means, methods and steps described in thespecification.

In keeping with long-standing patent law convention, the words “a” and“an” when used in the present specification in concert with the wordcomprising, including the claims, denote “one or more.” Some embodimentsof the invention may consist of or consist essentially of one or moreelements, method steps, and/or methods of the invention. It iscontemplated that any method or composition described herein can beimplemented with respect to any other method or composition describedherein.

The present disclosure concerns the production and use of therapeuticT-cells for individuals that are in need of HPV-specific T-cells, e.g.,HPV16- and/or HPV18-specific T-cells, including for treating HPVinfection and HPV-associated medical conditions. In particularembodiments, the methods and compositions are useful for treatingneoplasms that are indirectly or directly related to HPV infection, andsuch neoplasms may be benign or malignant. Between 13 and 18 HPV strainshave been characterized as conferring a high oncogenic risk, with 12 ofthese strains belonging to the HPV species 7 (HPV-18, -39, -45, -59,-68) and species 9 (HPV-16, -31, -33, -35, -52, -58, -67). HPV Types 6and 11 cause laryngeal papillomatisis.

I. HPV Antigen(s) and Generation of Pepmixes

Methods of the disclosure utilize antigen-presenting cells that presentmixtures of peptides to T-cells. Such “loaded” APCs are generated priorto exposure to peripheral blood T-cells for stimulation of theperipheral blood T-cells, and the generation of the loaded APCs may ormay not be performed by the individual or entity that performs thestimulation step for the peripheral blood T-cells. Thus, in someembodiments, an effective amount of a library of peptides is provided toAPCs as part of methods that ultimately generate therapeutic CTLs. Inmethods of the disclosure, prior to a stimulation step, APCs are exposedto a sufficient amount of the library of peptides. The library, inparticular cases, comprises a mixture of peptides (“pepmixes”) that spanpart or all of the same antigen, although in some cases the librarycomprises pepmixes that span part or all of one or more antigens, andthe one or more antigens may or may not be from the same HPV. Inparticular embodiments, peptides for the APCs are non-natural, and theymay or may not be chemically synthesized or produced by recombinantmeans.

In utilizing a library of mixtures of peptides from one or more HPVantigens, the various peptides may come from any part of a givenprotein, but in specific cases the peptides collectively span the lengthof the majority or all of the protein, wherein the sequence of thepeptides overlap at least in part to facilitate coverage of the entiredesired region of the specific antigen(s). In some cases the peptidesspan the length of one or more known epitopes or domains of therespective antigen to which the peptides correspond. Certain regions maybe covered by peptides that span the length of the region, including aregion such as a N-terminal domain, C-terminal domain, extracellulardomain, or intracellular domain, for example.

The antigens from which the peptides are derived may be antigens forHPVs that may be of any kind, but in specific embodiments the antigensare such that they allow for direction of cytotoxic T-cells toneoplasms, including cancers, associated with HPV infection. Inparticular embodiments, the peptides are derived from, or have sequencethat corresponds to, at least part of one or more antigens of at leastone HPV type, including HPV16 and/or HPV18. For example, in late stagecervical cancer, the HPV virus integrates into a tumor cell genome andloses all of its other genes except E6 and E7, so in some cases theseantigens are targeted. In embodiments wherein one would treat an earlierstage of cancer, such as before the virus integrated, one could utilizepeptides from antigens other than E6 and E7, including E5 and L1 and L2,for example. However, given that the two primary oncoproteins of highrisk HPV types are E6 and E7, in specific embodiments the sequence ofthe peptides are obtained from E6 and/or E7 from any HPV, but HPV16and/or HPV18, in particular. Peptides from any of antigens E1, E2, E3,E4, E5, E6, E7, L1, and/or L2 may be utilized in methods of thedisclosure.

In some cases, a pepmix library includes peptides corresponding to oneor more antigens from a single type of HPV virus, and those peptides mayor may not provide sequence coverage across the entire antigen(s) inquestion. In other cases, a pepmix library includes peptidescorresponding to one or more antigens from more than one HPV virus, andthose peptides may or may not provide sequence coverage across theentire antigen(s) in question. The pepmix may or may not be enriched forpeptides corresponding to one or more certain regions of one or morecertain antigens or corresponding to the entirety of one or more certainantigens.

Pepmixes utilized in the disclosure may be from commercially availablepeptide libraries or may be synthetically generated, for example.Examples of available libraries include those from JPT Technologies(Springfield, Va.) or Miltenyi Biotec (Auburn, Calif.). The skilledartisan, based on known sequences of HPV16 E6, HPV16 E7, HPV18 E6, andHPV18 E7, for example, would have sufficient information to be able togenerate peptides that correspond to their exemplary, respectivesequences. An example of sequence of the HPV16 E6 protein is availableat the National Center for Biotechnology Information's GenBank® databaseat GenBank® Accession No. AIQ82776.1 GI:688010703. An example ofsequence of the HPV16 E7 protein is at GenBank® Accession No. AIQ82814.1GI:688010789. An example of sequence of the HPV18 E6 protein is atGenBank® Accession No. AGU90423.1 GI:537801975. An example of sequenceof the HPV18 E7 protein is at GenBank® Accession No. AGU90424.1GI:537801976.

In particular embodiments, a library is comprised of peptides of acertain length that correspond to their respective antigens, although insome cases a library is comprised of a mixture of peptides with two ormore different lengths. The peptides may be of a certain length(s) andthey may overlap in sequence of a certain amount, although there may bevariability of length of overlap in some libraries. In particularembodiments, the peptides are at least 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,or 35 or more amino acids in length, for example. In particularembodiments, there is overlap among the peptides of at least 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, or 34 amino acids in length, forexample. In specific embodiments, the peptides are 15 amino acids longand overlap one another by 11 amino acids. A mixture of differentpeptides may include any ratio of the different peptides, although insome embodiments each particular peptide is present at substantially thesame numbers in the mixture as another particular peptide. Althoughcoverage of an antigen in sequence for the peptides may be random andsubstantially even over a given region of an antigen, in someembodiments a library may be enriched for one or more particularpeptides, such as one or more peptides that are known to encode anepitope or a part thereof, for example.

In particular embodiments, the pepmix for a particular antigen proteincomprise all possible HLA class I epitopes that are 8 to 10 amino acidslong, for example. In specific embodiments, longer peptides are utilizedto cover all class II epitopes for a particular peptide. In certainaspects, the peptides are at a maximum of 30 amino acids in length withoverlapping of 25 amino acids.

II. Methods of Producing and Using Therapeutic T-Cells

A. Producing Therapeutic T-Cells

In certain aspects, the present disclosure concerns the development ofimmune cells, such as cytotoxic T-cells, that target one or moreantigens from at least one HPV virus.

Methods disclosed herein may involve the stimulation and/or expansion ofimmune cells. The methods may involve the stimulation and/or expansionof peripheral blood cells, such as peripheral blood mononuclear cells.The methods may involve the stimulation and/or expansion of T cells. Thecells may have been obtained from the patient to be treated (i.e.,autologous cells), or from another individual (i.e., allogeneic cells).The methods involve stimulation and/or expansion of isolated immunecells, in certain embodiments. That is, specific methods may beperformed on a population of cells that contains substantially nonon-immune cells, such as erythrocytes. In some cases, the immune cellsare isolated PBMCs, or isolated T cells. The cells may have beenobtained from a sample of blood, such as a sample of blood obtained fromthe patient or individual. Certain methods disclosed herein involve astep of obtaining PBMCs and/or T cells from a sample obtained from thepatient. Certain embodiments of methods do not involve the step ofobtaining a sample of blood or cells from the patient or individual, butinstead are performed on a sample or cells that have been previouslyobtained. The method may involve processing the sample, such asenriching the sample for immune cells, such as PBMCs and/or T cells.Such methods may involve removing or substantially reducing the amountof, erythrocytes, platelets, serum and/or plasma in a sample. This mayresult in a population of immune cells containing substantially no othercells, such as erythrocytes. Methods disclosed herein may be performedon isolated immune cells, or a sample containing immune cells inaddition to other cells.

In methods of producing the T-cells, peripheral blood T-cells may beinitially stimulated with APCs that have been exposed to one or morepeptides that span some or all of at least one HPV antigen. Theantigenic peptides may be provided to the APCs in a library of peptidemixtures, and multiple libraries of pepmixes may be provided to the samecollection of APCs. In some embodiments, the collection includes bothimmunodominant and subdominant antigens.

In embodiments of the disclosure, therapeutic T-cells are generated andmay be provided to an individual that has an HPV infection or is at riskof having an HPV-associated medical condition that results indirectly ordirectly from an HPV infection. In methods of producing the therapeuticT-cells, under certain conditions peripheral blood T-cells are mixedwith APCs that are loaded with a library of peptides that span part orall of one or more antigens, including part or all of a HPV16 and/orHPV18 antigen, including E6 and/or E7, for example. In specificembodiments, for the stimulating step the T-cells reside within apopulation of PBMCs.

In some embodiments, the APCs used in certain steps may be dendriticcells (DCs). Methods for generation of DCs are well known in the art,e.g. see Ramos et al., supra. Monocytes may be isolated from PBMCs byCD14 selection and cultured in DC medium and 2 mM alanyl-glutamine with800 U/ml granulocyte/macrophage colony stimulating factor (GM-CSF) and1000 U/ml interleukin 4 (IL-4) for 5 days. GM-CSF and IL-4 may bereplenished on day 3. On day 5, DCs are matured in DC media with 10ng/ml interleukin-1β (IL-1β), 100 ng/ml interleukin 6 (IL-6), 10 ng/mlprostaglandin E2, 800 U/ml GM-CSF and 1000 U/ml IL-4. DC maturation maybe assessed by flow cytometry to detect upregulation of CD80. CD83, CD86and HLA-DR.

In some embodiments, the APCs used in certain steps are activatedT-cells. Activated T-cells may be polyclonal T-cells (T-APCs) generatedusing a portion of the autologous PBMC isolated from the venesectedblood. The cells may be activated by culturing in cell culture platesthat are coated with anti-CD3 and anti-CD28 antibodies. The cells arethen cultured to expand in the presence of IL-2 for 2 weeks. Theexpanded T-APC can be cryopreserved for later use. 2-3 days prior tousing T-APC for stimulation (e.g., for the 3rd cycle of stimulation andoptionally for subsequent stimulations), cryopreserved cells are thawedand re-stimulated in anti-CD3 and anti-CD28 antibody-coated cell cultureplates. On the day of stimulation, the T-APC cells are harvested andpulsed with the HPV E6/E7 peptides, followed by adding to the on-goingculture of HPV stimulated T-cells at 1:1 ratio.

In some embodiments, the APCs used in certain steps and/or methods maybe B-blasts (BBs). B-blasts may be generated from a patient's autologousPBMC, for example. The B lymphocytes within the PBMCs are activated byco-culturing with an irradiated allogeneic CD40L-expressing MRC5epithelial cell line and expanded in media containing 100 U/ml IL-4 and1 microgram/ml cyclosporin A.

In some embodiments, there is a method of generating T-cells that targetat least one antigen from one or both of HPV16 and HPV18, and thisoccurs generally by contacting a plurality of PBMCs with a plurality ofAPCs loaded for peptides from a library of peptides that correspond toone or more particular HPV16 and/or HPV18 viral antigens. In specificembodiments, the exposure of the two populations of cells allows forexpansion of the T-cells. In particular embodiments, the stimulationstep(s) occurs in the presence of one or more particular cytokines,which may be mammalian (e.g. murine, human) or human cytokines. Incertain embodiments, the one or more cytokines are IL-7 and IL-15,although in alternative embodiments the cytokine(s) are selected fromthe group consisting of IL-15, IL-7, IL-21, IL-12, IL-6, IL-4, and acombination thereof. In specific embodiments, one or more steps of themethods do not occur in the presence of IL-2, IL-4, IL-6, IL-7, IL-12,and/or IL-21, although alternatively IL-2, IL-4, IL-6, IL-7, IL-12,and/or IL-21 may be utilized. Reference to the presence of a cytokine isto presence of exogenously added cytokine, i.e. excluding any cytokinepresent within or secreted by the culture of cells. In some embodiments,the peptides are further defined as peptides that overlap in sequence tospan part or all of a HPV antigen. For example, in certain aspects thepeptides overlap by at least 10 amino acids, and particularly 11, and insome embodiments the peptides are at least 12 or more amino acids inlength, and particularly 15 amino acids in length.

The selection of an appropriate amount or concentration of a givencytokine for inclusion in a cell culture is within the ability of theperson or ordinary skill in the art. By way of example, the following isa list of certain interleukins and examples of appropriateconcentrations that may be used:

Interleukin 6 (IL-6): 50 to 150 ng/ml, one of about 50 ng/ml, 60 ng/ml,70 ng/ml, 80 ng/ml, 90 ng/ml, 100 ng/ml, 110 ng/ml, 120 ng/ml, 130ng/ml, 140 ng/ml or 150 ng/ml;

Interleukin 7 (IL-7): 5 to 15 ng/ml, one of about 5 ng/ml, 6 ng/ml, 7ng/ml, 8 ng/ml, 9 ng/ml, 10 ng/ml, 11 ng/ml, 12 ng/ml, 13 ng/ml, 14ng/ml or 15 ng/ml;

Interleukin 12 (IL-12): 5 to 15 ng/ml, one of about 5 ng/ml, 6 ng/ml, 7ng/ml, 8 ng/ml, 9 ng/ml, 10 ng/ml, 11 ng/ml, 12 ng/ml, 13 ng/ml, 14ng/ml or 15 ng/ml;

Interleukin 15 (IL-15): 5 to 15 ng/ml, one of about 5 ng/ml, 6 ng/ml, 7ng/ml, 8 ng/ml, 9 ng/ml, 10 ng/ml, 11 ng/ml, 12 ng/ml, 13 ng/ml, 14ng/ml or 15 ng/ml.

Table 1 below provides examples of certain embodiments of methods of thedisclosure.

TABLE 1 Examples of Elements of a Method Embodiments Examples forEmbodiments First Stimulation Source of T-cells Peripheral bloodmononuclear cells (PBMC) Non-adherent PBMC Antigen-presenting Dendriticcells (DC)s, PBMCs or B-blasts cells (APC) Cytokines Combinations ofIL-15 and IL-7, optionally with IL-6 and/or IL-12 and/or IL-21 and/orIL-4 Antigen Viral pepmixes for HPV Second stimulation Source of T-cellsProduct of first stimulation APCs DC PBMCs Autologous activated T-cells(AATC) Cytokines IL-15 and IL-7, preferably no IL-6 or IL-12 andoptionally IL-15 and IL-7 are the only interleukins Antigen Viralpepmixes for HPV Third stimulation Source of T-cells Product of secondstimulation (and subsequent stimulations as desired) APCs Pepmix-loadedAATCs + costimulatory cells Cytokines IL-15 and IL-7, preferably no IL-6or IL-12 and optionally IL-15 and IL-7 are the only interleukinsCostimulatory cells Cells expressing CD86, 4-1BB, and CD83, e.g., K562cells

Thus, in particular embodiments, a population of T-cells (wherein thepopulation may comprise substantially all T-cells or wherein thepopulation of T-cells is within another population of cells, such aswithin PBMCs) is exposed to a population of APCs to generate T celllines having particular characteristics, including at least: a)effectiveness at targeting HPV16 E6 and/or E7 and/or effectiveness attargeting HPV18 E6 and/or E7; b) polyclonality; c) TH1 bias; or d) acombination thereof. The generated T cell lines may be produced to beeffective at targeting HPV species 7 (HPV-18, -39, -45, -59, -68) andspecies 9 (HPV-16, -31, -33, -35, -52, -58, -67), and types 6 and 11,and this may be the results of pepmixes directed to any one or more ofthe following antigens: E1, E2, E3, E4, E5, E6, E7, L1, and/or L2.

In some cases, T-cells are stimulated more than once, and differentstimulation steps may or may not expose the population of cells to thesame conditions. In specific embodiments, a first stimulation hasconditions different from a subsequent stimulation, including a secondstimulation and/or a third stimulation. In specific embodiments, a firststimulation step of the method utilizes APCs that are pepmix-loaded DCsor pepmix-loaded PBMCs and utilizes IL-7 and IL-15. This stimulationstep may optionally be repeated one or more times.

In certain embodiments of the methods, between days 8 and 10 followingan initial exposure of the peripheral blood T-cells (or PBMCs) to thepepmix or APCs, there may be a re-stimulation of the PBMCs on day 8, day9, or day 10, but not later, and then a subsequent re-stimulation mayoccur on day 15, day 16, or day 17.

In a stimulation step that is subsequent to the first stimulation step(including optional repeats of the first stimulation step), theresultant T-cells obtained after the first stimulation (and which may bein a heterogeneous population of cells) are exposed to pepmix-loaded DCsor pepmix-loaded PBMCs and/or autologous activated T-cells. In astimulation that is subsequent to first and second stimulation steps,T-cells obtained after the second or later stimulation (and which mayreside in a heterogeneous population of cells) are exposed topepmix-autologous activated T-cells. Costimulatory cells that may beutilized in any stimulation step include at least cells that expressCD86, 4-1BB, CD83, CD40, OX40, and/or CD80. In specific cases, thecostimulatory cells may be K562 cells.

In some embodiments, during the steps of the method the cells in cultureare modified. In specific embodiments, the cells are modified to harbora polynucleotide that expresses a gene product that renders the cellseffective or more effective for a specific purpose or function, such aseffective or more effective for targeting a particular target and/orenhanced in function for T-cell-mediated cytotoxicity, and/or modifiedto resist tumor antigen-specific cellular immunity, for example.

In some embodiments, the cells are modified to express a certainnon-natural receptor that allows the T-cells to effectively or moreeffectively target a desired target cell, such as one that expresses acertain antigen. The cells may be modified to express a chimeric antigenreceptor (CAR), an αβ T-cell receptor, and so forth. The cells may bemodified to express an expression vector (that may be viral (includingretroviral, lentiviral, adenoviral, adeno-associated viral, and soforth) or non-viral) during the method at specific time points, such asthe vector being introduced between day 2 and 5 of culture, for example.In some embodiments the cells are exposed to the expression vectorwithin about 3 days after each stimulation, but in such cases themodification occurs in more differentiated T-cells that have less longterm potential (which in specific circumstances is desirable).

In specific embodiments, the cells are modified to express a CAR thattargets a cancer antigen, such as EphA2, HER2, GD2, Glypican-3, 5T4,8H9, α_(v)β₆ integrin, B cell maturation antigen (BCMA) B7-H3, B7-H6,CAIX, CA9, CD19, CD20, CD22, kappa light chain, CD30, CD33, CD38, CD44,CD44v6, CD44v7/8, CD70, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFRvIII,EGP2, EGP40, EPCAM, ERBB3, ERBB4, ErbB3/4, FAP, FAR, FBP, fetal AchR,Folate Receptor α, GD2, GD3, HLA-AI MAGE A1, HLA-A2, IL11Ra, IL13Ra2,KDR, Lambda, Lewis-Y, MCSP, Mesothelin, Muc1, Muc16, NCAM, NKG2Dligands, NY-ESO-1, PRAME, PSCA, PSC1, PSMA, ROR1, Sp17, SURVIVIN, TAG72,TEM1, TEM8, VEGRR2, carcinoembryonic antigen, HMW-MAA, VEGF receptors,and/or other exemplary antigens that are present with in theextracellular matrix of tumors, such as oncofetal variants offibronectin, tenascin, or necrotic regions of tumors and othertumor-associated antigens or actionable mutations that are identifiedthrough genomic analysis and or differential expression studies oftumors, for example.

In some embodiments the cells are modified to resist tumorantigen-specific cellular immunity, e.g. mediated by transforming growthfactor beta (TGF-β). For example, the cells may be modified to express adominant negative receptor for TGF-beta (DNRII), e.g. as described inFoster et al., (Antitumor activity of EBV-specific T lymphocytestransduced with a dominant negative TGF-beta receptor. J Immunother.2008; 31:500-505, incorporated herein by reference). This may comprisetransfecting the cells with a retroviral expression vector encoding adominant negative TGF-β type II receptor (DNRII) modified by removal ofthe immunogenic hemagglutinin tag. Such modified T-cells have been shownto have a functional advantage over unmodified T-cells in the presenceof TGF-β-secreting tumor, including enhanced antitumor activity (Fosteret al., supra).

Methods according to the present invention may improve the rate ofexpansion for populations of virus-specific T-cells as compared to priorart methods. The rate of expansion for a T-cell population can beanalysed by methods well known to the skilled person. Methods includemeasuring the number of T-cells at one or more time points. For example,the number of T-cells can be determined after performing a methodaccording to the invention and compared to the number of T-cells at thebeginning of the method; fold expansion in the number of T-cells canthen be calculated.

Rates of expansion can also be determined by analysing cell division byT-cells over a period of time. Cell division for a given population ofT-cells can be analysed, for example, by in vitro analysis ofincorporation of ³H-thymidine or by CFSE dilution assay, e.g. asdescribed in Fulcher and Wong, Immunol Cell Biol (1999) 77(6): 559-564,hereby incorporated by reference in entirety.

The improvement in the rate of expansion achieved by the methodsaccording to the present invention can be determined by performing amethod according to the invention, and comparing the expansion forT-cells in that method to a comparable, control method, e.g. as per themethod of Ramos et al., (J Immunother 2013; 36:66-76).

In some embodiments, the rate of expansion for a population of T-cellsin a method according to the present invention is one of at least 1.001times, 1.002 times, 1.003 times, 1.004 times, 1.005 times, 1.006 times,1.007 times, 1.008 times, 1.009 times, 1.01 times, 1.02 times, 1.03times, 1.04 times, 1.05 times, 1.06 times, 1.07 times, 1.08 times, 1.09times, 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times,1.7 times, 1.8 times, 1.9 times, or 2 times the rate of expansion in acomparable control method.

The rate of expansion may be of the virus-specific T-cell population, orthe total T-cell population.

The virus-specific T-cells generated/expanded according to the method ofthe present invention may at least retain the same functional propertiesas virus-specific T-cells generated/expanded according to prior artmethods. That is, the accelerated rate of expansion does not negativelyinfluence the functional properties of the expanded T-cells.

For example, in embodiments wherein the methods generate/expand apopulation of virus-specific T-cells, the T-cells display similarcytotoxicity to cells infected with or comprising/expressing a peptideof the virus as virus-specific T-cells expanded according to prior artmethods.

Cytotoxicity of expanded T-cells can be analysed e.g. by culturing theexpanded T-cell population with APCs presenting a peptide of the virusfor which the T-cell is specific at different effector (i.e. T-cell) totarget (i.e. APC) ratios, and measuring specific lysis of the APCs. Forexample, cytotoxicity of an HPV-specific CTL population can be analysedby measuring specific lysis of HPV-transformed LCL cells at differenteffector to target ratios.

B. Using Therapeutic T-Cells

In certain embodiments, cells produced by methods of the disclosure areprovided to an individual in need thereof for treatment of a medicalcondition, including one caused by a viral infection, or to target aviral infection in which no symptoms of a medical condition aredetectable or have manifested. As used herein “treatment” or “treating,”includes any beneficial or desirable effect on the symptoms or pathologyof a disease or pathological condition, and may include even minimalreductions in one or more measurable markers of the disease or conditionbeing treated, e.g., cancer. Treatment can involve optionally either thereduction or amelioration of symptoms of the disease or condition, orthe delaying of the progression of the disease or condition. “Treatment”does not necessarily indicate complete eradication or cure of thedisease or condition, or associated symptoms thereof.

In the methods encompassed by the disclosure, the therapeutic T-cellsare utilized to treat viral-associated disease caused directly orindirectly by a single non-HPV virus or are otherwise provided to anindividual that is seropositive for a single non-HPV virus. In othercases, the therapeutic T-cells are utilized to treat viral-associateddisease(s) caused directly or indirectly by more than one virus or areotherwise provided to an individual that is seropositive for more thanone virus. In the collection of therapeutic T-cells, each T-cell and itsprogeny has specificity for only one peptide in one antigen from onevirus, and upon production of the collection of therapeutic T-cells, oneexpands a population of T-cell clones that together havemulti-specificity, such as for multiple epitopes in each viral antigen,for example.

In at least some methods of the disclosure, a therapeutically effectiveamount of the CTLs generated thereby are administered to an individual,for example, an individual known to have or suspected of having orsusceptible to having HPV16 and/or HPV18-associated disease. In specificembodiments, the cells are administered by injection, such asintravenous, intramuscular, intradermal, subcutaneous, intraperitonealinjection, and so forth, for example. In some embodiments, the CTLs arefurther defined as polyclonal CD4+ and CD8+ CTLs. The PBMCs may beallogeneic to the individual or may be autologous to the individual.

In certain cases, neoplasms are treated with cells of the disclosure,and the neoplasm may be benign, malignant, or a premalignant lesion thatcan lead to cancer. Thus, an individual may be treated with cellsproduced by methods of the disclosure at the premalignant lesion stageand/or after the lesion becomes malignant. The individual may have earlyor late stage cancer, and the skilled artisan is aware that the methodsof producing the cells may be tailored for such different stages ofcancer, such as by utilizing peptides for the APCs that are fromantigens associated with early vs. late stage cancer. In specificembodiments, the cancer may be primary, metastatic, recurrent,refractory, and so forth.

In certain cases, premalignant lesions that can lead to cancers, such aspremalignant lesions of the cervix, vulva, vagina, penis, larynx,oropharynx anus, and other upper aerodigestive areas, for example, aretreated with cells produced by methods of the disclosure. Thus, anindividual may be treated with cells produced by methods of thedisclosure at the premalignant lesion stage and/or after the lesionbecomes malignant. HPV-associated medical conditions that may be treatedwith cells produced by methods of the disclosure include at leastdysplasias of the genital area(s), cervical intraepithelial neoplasia,vulvar intraepithelial neoplasia, penile intraepithelial neoplasia, analintraepithelial neoplasia, cervical cancer, anal cancer, vulvar cancer,vaginal cancer, penile cancer, genital cancers, oropharyngeal cancer,nasopharyngeal carcinoma, oral papillomas and other upper aerodigestivelesions.

In some cases, one can determine the serotype that is associated with acancer before administration of the cells, although in some cases theserotype is not determined. In specific embodiments, HPV16-specific orHPV18-specific cells have activity for tumors that are HPV16 orHPV18-positive, respectively, although in some cases there iscross-reactivity with different HPV serotypes. The ability tocross-react may or may not be known, and in certain cases, for example,an individual with HPV16 infection or HPV16-associated medical conditionis administered HPV18-specific T-cells, and vice versa. In such cases,an individual may be treated with cells specific for a serotype in whichit is unknown if the individual has that serotype, yet the cells stillare therapeutically effective because of cross-reactivity.

In cases wherein the APCs of the stimulation steps of the method areloaded with HPV16 and HPV18 pepmixes together, the outcome ofadministration of T-cells expanded through such APCs is determined bywhether the individual has been exposed to the virus in question. Forexample, if an individual is infected with HPV18 and not HPV16, onlyHPV18-specific T-cells will respond, and this is because the infectionwill initially have stimulated a T-cell response to HPV 18. ThoseT-cells will expand in the individual and then become memory T-cells andwould be at higher numbers than T-cells specific for HPV16 that havenever been activated, for example.

The individual being treated may be known to have cancer, suspected ofhaving cancer, or at risk for having cancer (such as personal or familyhistory; being sexual active, including sexually promiscuous; and/orhaving a genetic predisposition, including one or more specificmarkers). An individual being treated may have the presence of the HPVvirus but there are not yet any deleterious symptoms of a HPV-relatedmedical condition. The individual may have a benign or malignantneoplasm. The individual may have early or late stage cancer, and theskilled artisan is aware that the methods of producing the cells may betailored for such different stages of cancer, such as by utilizingpeptides for the APCs that are from antigens associated with early vs.late stage cancer. In specific embodiments, the HPV-associated diseaseis malignant cancer of the mouth or genital region. In specificembodiments, the cancer may be primary, metastatic, recurrent,refractory, and so forth. The individual may be infected with HPV16and/or HPV18 as a result of sexual acts of any kind or intimate physicalcontact of any kind.

Any stage of HPV infection may be treated with cells encompassed by thedisclosure. The individual with established HPV-associated cancer beingtreated with methods of the disclosure include Carcinoma in Situ (Stage0), Stage I, Stage II, Stage III, or Stage IV (which may be determinedby MRI, CT scan, PET scan, etc.). Additionally, individuals withpre-cancer lesions (dysplasia) may also be treated.

In some embodiments, one or more administrations of the cells producedby methods of the disclosure are provided to an individual in needthereof. The length of time between different administrations may be ofany suitable duration, including on the order of 1-7 days, 1-4 weeks,1-12 months, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more years. Multipleinfusions within about a year may be employed, in some cases. In caseswherein more than one administration of cells are provided to theindividual, the antigen to which the cells are targeted may or may notbe the same antigen that was targeted with the cells utilized in earlieradministration(s). For example, in a first administration of cells, thecells may target HPV18 E6, whereas in another administration of cells,the cells target HPV18 E7, or vice versa. Additional administration(s)may be required in cancers that become refractory, for example.Additional stimulations may be employed in conjunction with one or moreother types of cancer treatments.

In some cases, an individual is optionally determined to have HPVinfection by any suitable means in the art. Because HPV cannot becultured in cell cultures, one may utilize HPV infection diagnosismethods such as DNA tests utilizing PCR, Southern blot hybridization,and/or in situ hybridization, and these methods may or may not be usedin conjunction with colposcopy; acetic acid test; biopsy; physicalexamination; and/or Pap smear, for example.

In specific embodiments, a male individual is provided an effectiveamount of cells produced by methods of the disclosure to target HPV withwhich he is infected, and in such a case the individual thereafter has areduced chance of infecting another, such as a female individual throughsexual activity. The male individual may or may not be determined to beinfected with HPV prior to exposure to the cells of the methods of thedisclosure. In some cases, if an individual is shown to be infected withan oncogenic HPV, it would be worth treating him with cells to eliminatehis risk. If the cells were effective, they would also reduce thechances of him transmitting the virus to his partner.

In specific embodiments, the individual is immunocompromised (which forexample, may be defined as an individual whose ability to fightinfectious disease or cancer with the immune system is compromised orentirely absent). In specific embodiments, the immunocompromisedindividual has had a stem cell transplant (including hematopoietic stemcell transplantation), has had an organ transplant and/or has receivedone or more cancer treatments, including chemotherapy or radiation, forexample. In some cases, the individual has acquired or inherited immunedeficiency disorder. In some embodiments, those that areimmunocompromised by their disease and/or its treatment are providedmethods and/or compositions of the disclosure.

Methods of medical treatment may involve treatment of cancer by a methodof ameliorating, treating, or preventing a malignancy in a human subjectwherein the steps of the method assist or boost the immune system ineradicating cancerous cells. Such methods may include the administrationof cells, according to the present invention that invoke an active (orachieve a passive) immune response to destroy cancerous cells. Methodsof treatment may optionally include the co-administration of biologicaladjuvants (e.g., interleukins, cytokines, Bacillus Comette-Guerin,monophosphoryl lipid A, etc.) in combination with conventional therapiesfor treating cancer such as chemotherapy, radiation, or surgery. Methodsof treatment may involve administering a composition according to thepresent invention as a vaccine that works by activating the immunesystem to prevent or destroy cancer cell growth. Methods of medicaltreatment may also involve in vivo, ex vivo, and adoptiveimmunotherapies, including those using autologous and/or heterologouscells or immortalized cell lines.

III. Pharmaceutical Compositions

In accordance with this disclosure, the term “pharmaceuticalcomposition” relates to a composition for administration to anindividual. In a particular embodiment, the pharmaceutical compositioncomprises a composition comprising therapeutic immune cells forparenteral, transdermal, intraluminal, intra-arterial, intrathecal orintravenous administration or for direct injection into a neoplasm, suchas a cancer. It is in particular envisaged that the pharmaceuticalcomposition is administered to the individual via infusion or injection.Administration of the suitable compositions may be effected by differentways, e.g., by intravenous, subcutaneous, intraperitoneal,intramuscular, topical or intradermal administration.

The pharmaceutical composition of the present disclosure may furthercomprise a pharmaceutically acceptable carrier. Examples of suitablepharmaceutical carriers are well known in the art and include phosphatebuffered saline solutions, water, emulsions, such as oil/wateremulsions, various types of wetting agents, sterile solutions, etc.Compositions comprising such carriers can be formulated by well-knownconventional methods. These pharmaceutical compositions can beadministered to the subject at a suitable dose.

The dosage regimen will be determined by the attending physician andclinical factors. As is well known in the medical arts, dosages for anyone patient depends upon many factors, including the patient's size,body surface area, age, the particular compound to be administered, sex,time and route of administration, general health, and other drugs beingadministered concurrently. A particular dosage for administration mightbe in the range of 2×10⁷ cells per m² to 1×10¹⁰ cells per m² of bodysurface area. Progress can be monitored by periodic assessment.

The compositions of the disclosure may be administered locally orsystemically. In a preferred embodiment, the pharmaceutical compositionis administered subcutaneously and in an even more preferred embodimentintravenously. Preparations for parenteral administration includesterile aqueous or non-aqueous solutions, suspensions, and emulsions.Examples of non-aqueous solvents are propylene glycol, polyethyleneglycol, vegetable oils such as olive oil, and injectable organic esterssuch as ethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishes,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like. In addition, the pharmaceutical composition of thepresent disclosure might comprise proteinaceous carriers, like, e.g.,serum albumin or immunoglobulin, preferably of human origin. It isenvisaged that the pharmaceutical composition of the disclosure mightcomprise, in addition to the cells as described in this disclosure,further biologically active agents, depending on the intended use of thepharmaceutical composition.

IV. Combination Therapy

In certain embodiments of the disclosure that concern CTLs generatedagainst HPV antigen(s), methods of the present disclosure for clinicalaspects are combined with other agents effective in the treatment ofhyperproliferative disease, such as anti-cancer agents. An “anti-cancer”agent is capable of negatively affecting cancer in a subject, forexample, by killing cancer cells, inducing apoptosis in cancer cells,reducing the growth rate of cancer cells, reducing the incidence ornumber of metastases, reducing tumor size, inhibiting tumor growth,reducing the blood supply to a tumor or cancer cells, promoting animmune response against cancer cells or a tumor, preventing orinhibiting the progression of cancer, or increasing the lifespan of asubject with cancer. More generally, these other compositions may beprovided in a combined amount effective to kill or inhibit proliferationof the cell. This may be achieved by contacting the cancer cell with asingle composition or pharmacological formulation that includes bothagents, or by contacting the cancer cell with two distinct compositionsor formulations, at the same time, wherein one composition includes theexpression construct and the other includes the second agent(s). Inother cases, administration of the cells and a second composition may beseparate and may have separate administration routes and/or carriers.

Tumor cell resistance to chemotherapy and radiotherapy agents representsa major problem in clinical oncology. One goal of current cancerresearch is to find ways to improve the efficacy of chemo- and/orradiotherapy by combining it with additional therapy. In the context ofthe present disclosure, it is contemplated that cell therapy could beused similarly in conjunction with chemotherapeutic, radiotherapeutic,and/or immunotherapeutic intervention, for example.

Alternatively, the present inventive therapy may precede or follow theother agent treatment by intervals ranging from minutes to weeks,months, or years. In embodiments where the other agent and presentinvention are applied separately to the individual, one would generallyensure that a significant period of time did not expire between the timeof each delivery, such that the agent and inventive therapy would stillbe able to exert an advantageously combined effect on the cell. In suchinstances, it is contemplated that one may contact the cell with bothmodalities within about 12-24 h of each other and, more preferably,within about 6-12 h of each other. In some situations, it may bedesirable to extend the time period for treatment significantly,however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2,3, 4, 5, 6, 7 or 8) lapse between the respective administrations.

It is expected that the treatment cycles would be repeated as necessary.It also is contemplated that various standard therapies, as well assurgical intervention, may be applied in combination with the inventivecell therapy.

Examples of HPV-associated cancer treatments (as an example) that may beused in conjunction with cells produced from methods of the disclosureinclude at least the following: 1) surgery (tumor resection, neckdissection, conization, hysterectomy, and so forth); 2) drug therapythat may include Avastin® (Bevacizumab); Blenoxane (Bleomycin);Hycamtin® (Topotecan Hydrochloride); or a combination thereof; 3)radiotherapy; 4) immunotherapy other than that of the disclosure; 5)hormone therapy; or 6) a combination thereof.

V. Kits of the Disclosure

Any of the compositions described herein may be comprised in a kit. In anon-limiting example, a library of pepmixes may be comprised in a kit,any type of cells may be provided in the kit, and/or reagents formanipulation of pepmixes and/or cells may be provided in the kit.Cytokine(s) or means of producing them (such as vectors that encodethem) may be included in the kit. Cell culture reagents and/orapparatus(es) may be included. The component(s) are provided in suitablecontainer means.

In one embodiment a kit may comprise a container comprising a quantityof T-cells obtained by a method of the present invention formulated foradministration to a subject (e.g. by admixture with a suitable carrier,excipient, diluent, or adjuvant) preferably by infusion, more preferablyfor administration by infusion in a method of autologous adoptivecellular immunotherapy. The kit may be maintained at a predeterminedtemperature, e.g. less than about 4° C., less than about −2° C. or lessthan about −50° C. The kit may further comprise instructions for thestorage and/or transport of the kit and/or for the administration of theT-cells.

The kits may comprise a suitably aliquoted compositions of the presentinvention. The components of the kits may be packaged either in aqueousmedia or in lyophilized form. The container means of the kits willgenerally include at least one vial, test tube, flask, bottle, syringeor other container means, into which a component may be placed, andpreferably, suitably aliquoted. Where there are more than one componentin the kit, the kit also will generally contain a second, third or otheradditional container into which the additional components may beseparately placed. However, various combinations of components may becomprised in a vial. The kits of the present invention also willtypically include a means for containing the components in closeconfinement for commercial sale. Such containers may include injectionor blow molded plastic containers into which the desired vials areretained.

However, the components of the kit may be provided as dried powder(s).When reagents and/or components are provided as a dry powder, the powdercan be reconstituted by the addition of a suitable solvent. It isenvisioned that the solvent may also be provided in another containermeans.

In some cases, reagents and/or devices to detect HPV infection may beincluded in the kit. Examples include swabs, spatulas, cytobrushes,slides, cover slips, cytology sample collection receptacle(s), and soforth. Additional drugs for HPV infection or cancer may be included inthe kit, such as Bevacizumab; Bleomycin; Topotecan Hydrochloride; or acombination thereof.

EXAMPLES

The following examples are presented in order to more fully illustratethe preferred embodiments of the invention. They should in no way,however, be construed as limiting the broad scope of the invention.

Example 1 Production of Therapeutic T-Cells

In some embodiments of the disclosure, there is a mechanism by which onecan rapidly generate a single preparation of T-cells, includingpolyclonal (for example, CD4+ and CD8+) CTLs, that are consistentlyspecific for a variety of antigens derived from one or more humanpapillomaviruses that can prove fatal. The disclosure is readilyadaptable to clinical implementation and can be used as an “off theshelf” HPV antiviral agent. The methods and compositions are readilyadaptable to clinical implementation and are useful as a safe andeffective HPV antiviral agent for individuals.

In specific embodiments, peripheral blood T-cells were stimulated withmonocyte-derived dendritic cells loaded with pepmixes [peptide librariesof 15-mers overlapping by 11 amino acids (aa)] spanning E6/E7, in thepresence or absence of specific accessory cytokines. The resultingT-cell lines were further expanded with pepmix-loaded activated B-cellblasts. There was successfully reactivation and expansion (>1200-fold)of E6-specific/E7-specific T-cells from 8/16 cervical and 33/52oropharyngeal cancer patients.

The presence of the cytokines interleukin (IL)-6, IL-7, IL-12, and IL-15is useful in the method, in specific embodiments of the methods. Theproduced T-cell lines possess the desirable characteristics ofpolyclonality, multiple T-cell subset representation (including thememory compartment) and a TH1 bias, and eliminate E6/E7 targets. Thedisclosure has shown that it is possible to robustly generate HPV16E6/E7-directed T-cell lines from patients with HPV16-associated cancers.Because the technique is scalable and good-manufacturingprocedures-compliant, these lines are useful for adoptive cellularimmunotherapy of patients with HPV16 cancers and may be applied to HPV18cancers also.

Known methods for producing T-cells for HPV16 are demonstrated in FIG.1A, showing results for 3 HPV-associated cancer patients (γIFN ELISpotassay obtained in cell lines obtained after stimulation of PBMCs by DCsloaded with only HPV16-pepmix). In FIG. 1B, results are shown for 2 ofthe patients whose results are also demonstrated in FIG. 1A, in additionto a third individual. FIG. 1B shows results of γIFN ELISpot assay forcell lines obtained after stimulation of PBMCs by DCs loaded withHPV16-pepmix and HPV18-pepmix. Reactivity against both HPV16 and HPV18antigens can be detected (not all patients will have reactivity againstboth serotypes).

Turning to specifics of the methods, in certain cases DCs are loadedwith HPV16-E6/E7 and HPV18-E6/E7 pepmix libraries. In such cases, thecell lines are able to recognize both HPV16 and HPV18 E6 and E7 antigens(instead of only HPV16 antigens, for example). In at least certaincases, expansion of the T-cells occurs in the presence of IL-7 and IL-15but not IL-2. The presence of IL-7 and IL-15 in conditions for themethod may or may not be at each step of stimulation and expansion. Insome embodiments, expansion of the HPV-specific T-cells after initialgeneration/expansion with DCs occurs not with autologous B-blasts loadedwith pepmixes in the presence of IL-15 but instead utilizes autologous,polyclonal activated T-cells loaded with pepmix, in the presence ofcostimulatory cells (CD80/CD86/CD83/4-1BBL), and IL-7 and IL-15.Employing these conditions, T-cell expansion occurs at a more rapidrate, at least 10-fold as that obtained by known methods, withsuccessful demonstration having occurred after 3 rounds of stimulationand without loss of specificity.

Summary of fold cellular expansion using the known method with 3HPV-associated cancer patients is provided in Table 2.

TABLE 2 Fold expansion at the end of each stimulation with known methodAfter 1^(st) stimulation After 2^(nd) stimulation After 3^(rd)stimulation Patient (with DC and IL- (with DC and IL- (with B-blasts ID2/15) 2/15) and IL-2/15) OPA 3.38 2.63 4.96 OPE 1.59 4.60 1.60 OPY 2.202.50 0.38

A summary of fold cellular expansion using a novel method of thedisclosure with 3 HPV-associated cancer patients is shown in Table 3.Fold expansion after 3 rounds of stimulation is on average approximately50 times higher than using a known method. Specificity is maintained(illustrated in #1).

TABLE 3 Fold expansion at the end of each stimulation with a method ofthe disclosure After 3^(rd) stimulation After 1^(st) stimulation After2^(nd) stimulation (with activated T- Patient (with DC and IL- (with DCand IL- cells, costim cells ID 7/15) 7/15) and IL-7/15) PDC 2.60 5.35151.20 PGD 5.00 6.36 120.00 PJK 3.58 5.53 60.00

Example 2 Protocol for the Expansion of HPV T-Cells

HPV stimulated T-cells (HPVST) are first activated by HPV E6/E7peptide-pulsed autologous dendritic cells (DCs) at 10-20:1 PBMC:DCratio, and cultured for 8 days in culture medium containing IL-6 (100ng/ml), IL-7 (10 ng/ml), IL-12 (10 ng/ml), IL-15 (10 ng/ml) (e.g. perthe first stimulation step described by Ramos et al., (J Immunother2013; 36:66-76)).

A second stimulation step on day 9 is carried out using peptide-pulsedDCs at 5-10:1 PBMC:DC ratio in media containing IL-7 (10 ng/ml) andIL-15 (100 ng/ml).

Subsequent weekly stimulation/expansion steps are then carried toachieve a desired number of HPVSTs out using HPV E6/E7 peptide-pulsedautologous T-APC at 1:1 ratio, in the presence of equal number ofirradiated allogeneic K562-cs co-stimulatory cells, and in mediacontaining IL-7 (10 ng/ml) and IL-15 (100 ng/ml). The polyclonal T cells(T-APCs) are generated using a portion of the autologous PBMC isolatedfrom the venesected blood. The cells are activated by culturing in cellculture plates that are coated with anti-CD3 and anti-CD28 antibodies.The cells are then cultured to expand in the presence of IL-2 for 2weeks. The expanded T-APC can be cryopreserved for later use. 2-3 daysprior to using T-APC for HPVST re-stimulation (3rd cycle of HPVSTre-stimulation and onward), cryopreserved cells are thawed andre-stimulated in anti-CD3 and anti-CD28 antibody-coated cell cultureplates. On the day of HPVST re-stimulation, the T-APC cells areharvested and pulsed with the HPV E6/E7 peptides, followed by adding tothe on-going culture of HPVST at 1:1 ratio.

Example 3 In Vivo Expansion and Persistence of Infused HPVSTs in HumanPatients

HPVSTs obtained from Example 2 were transduced with a dominant negativereceptor for TGF-beta (DNRII) [see Foster et al., Antitumor activity ofEBV-specific T lymphocytes transduced with a dominant negative TGF-betareceptor. J Immunother. 2008; 31:500-505].

The HPVSTs were administered to two human patients and tested for invivo expansion and persistence. Patient #1 had widely metastaticoropharyngeal cancer and had discontinued prior therapy. Patient #2 hadoropharyngeal cancer metastatic to the neck and was receivingconcomitant nivolumab treatment, without prior response to nivolumabalone.

In vivo expansion and persistence of infused HPVSTs was assessed by qPCRfor the DNRII gene performed in PBMCs isolated from peripheral bloodfrom the patient. Data points in FIGS. 2 and 3 represent criticalpost-infusion intervals after the infusion of HPVSTs.

In patient #1 progressive expansion was observed (FIG. 2). In patient #2although expansion was limited (FIG. 3), with a peak at 6 weekscoinciding with re-infusion of HPVSTs, the patient had a partialclinical response. Six weeks after HPVST infusion patient #2 exhibited adecrease in disease burden measured by PET scan and physical examinationcompared to a pre-treatment baseline (FIG. 4).

Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the spirit andscope of the invention as defined by the appended claims. Moreover, thescope of the present application is not intended to be limited to theparticular embodiments of the process, machine, manufacture, compositionof matter, means, methods and steps described in the specification. Asone of ordinary skill in the art will readily appreciate from thedisclosure of the present invention, processes, machines, manufacture,compositions of matter, means, methods, or steps, presently existing orlater to be developed that perform substantially the same function orachieve substantially the same result as the corresponding embodimentsdescribed herein may be utilized according to the present invention.Accordingly, the appended claims are intended to include within theirscope such processes, machines, manufacture, compositions of matter,means, methods, or steps.

What is claimed is:
 1. A method of treating a human papillomavirus(HPV)-positive cancer in a subject, the method comprising: (1)generating or expanding a population of T cells specific for a HPV by amethod comprising: (a) obtaining peripheral blood T-cells from a subjectwith HPV-positive cancer; and (b) stimulating the peripheral bloodT-cells with antigen presenting cells in the presence of interleukin(IL)-7 and IL-15 and in the absence of IL-6 and/or IL-12, wherein theantigen presenting cells were previously exposed to one or morepeptides, wherein the peptides comprise sequence that corresponds to atleast 8 contiguous amino acids of the sequence of one or both of E6 andE7 of HPV; and (2) administering the generated or expanded population ofT cells to a subject with HPV-positive cancer.
 2. The method of claim 1,wherein after (1)(a) and before (1)(b) the method further comprisesstimulating the peripheral blood cells with antigen presenting cells inthe presence of IL-7 and IL-15, and in the presence of IL-6 and/orIL-12, wherein the antigen presenting cells were previously exposed toone or more peptides, wherein the peptides comprise sequence thatcorresponds to at least 8 contiguous amino acids of the sequence of oneor both of E6 and E7 of HPV.
 3. The method of claim 1, wherein after (1)and before (2) the method further comprises stimulating the populationof T-cells obtained from (1) with antigen presenting cells in thepresence of IL-7 and IL-15, and in the presence of co-stimulatory cells,wherein the antigen presenting cells were previously exposed to one ormore peptides, wherein the peptides comprise sequence that correspondsto at least 8 contiguous amino acids of the sequence of one or both ofE6 and E7 of HPV.
 4. The method of claim 3 wherein the co-stimulatorycells are CD80+, CD86+, CD83+, 4-1BBL+, CD40+ cells, OX40+ cells, or acombination thereof.
 5. The method of claim 1, wherein after (1) andbefore (2) the method further comprises (i) re-stimulating thepopulation of T-cells obtained from (1) in the presence of IL-7 andIL-15 but not in the presence of co-stimulatory cells, and (ii)stimulating the T-cells obtained after (i) with antigen presenting cellsin the presence of IL-7 and IL-15, and in the presence of co-stimulatorycells, wherein the antigen presenting cells were previously exposed toone or more peptides, wherein the peptides comprise sequence thatcorresponds to at least 8 contiguous amino acids of the sequence of oneor both of E6 and E7 of HPV.
 6. The method of claim 1, wherein theantigen presenting cells are dendritic cells (DC), B-blasts (BB), orperipheral blood mononuclear cells (PBMCs).
 7. The method of claim 1,wherein the cancer is cervical cancer, anal cancer, vulvar cancer,vaginal cancer, penile cancer, oropharyngeal cancer, nasopharyngealcarcinoma, laryngeal papillomatosis, laryngeal cancer, head and neckcancer, or a dysplasia of any of site thereof.
 8. A method of treating aHPV-positive cancer in a subject, the method comprising: (1) generatingor expanding a population of T cells specific for a HPV by a methodcomprising: (i) (a) obtaining peripheral blood T-cells from a subjectwith HPV-positive cancer; and (b) stimulating the peripheral bloodT-cells with antigen presenting cells in the presence of interleukin(IL)-7 and IL-15, wherein the antigen presenting cells were previouslyexposed to one or more peptides, wherein the peptides comprise sequencethat corresponds to at least 8 contiguous amino acids of the sequence ofone or both of E6 and E7 of HPV; (ii) stimulating the peripheral bloodT-cells obtained from (i) with antigen presenting cells in the presenceof interleukin (IL)-7 and IL-15 and in the absence of IL-6 and/or IL-12,wherein the antigen presenting cells were previously exposed to one ormore peptides, wherein the peptides comprise sequence that correspondsto at least 8 contiguous amino acids of the sequence of one or both ofE6 and E7 of HPV, wherein (ii) is optionally repeated one or more times;and (iii) stimulating T-cells obtained from (ii) with antigen presentingcells in the presence of interleukin (IL)-7 and IL-15, and in thepresence of co-stimulatory cells, wherein the antigen presenting cellswere previously exposed to one or more peptides, wherein the peptidescomprise sequence that corresponds to at least 8 contiguous amino acidsof the sequence of one or both of E6 and E7 of HPV, wherein (iii) isoptionally repeated one or more times; (2) administering the generatedor expanded population of T cells to a subject with HPV-positive cancer.9. The method of claim 8, wherein stimulation of T-cells in (i) is inthe presence of IL-6 and/or IL-12.
 10. The method of claim 8, whereinthe antigen presenting cells used in (i) and (ii) are dendritic cells(DC), B-blasts (BB), or peripheral blood mononuclear cells (PBMCs). 11.The method of claim 8, wherein the antigen presenting cells used in(iii) are activated T cells, dendritic cells (DC), B-blasts (BB), orperipheral blood mononuclear cells (PBMCs).
 12. The method of claim 8,wherein the co-stimulatory cells are CD80+, CD86+, CD83+, 4-1BBL+, CD40+cells, OX40+ cells or a combination thereof.
 13. A method of treating acancer in a HPV-positive subject, the method comprising: (1) generatingor expanding a population of T cells specific for a HPV by a methodcomprising: (a) obtaining peripheral blood T-cells from a subject withHPV-positive cancer; and (b) stimulating T-cells specific for HPV or foran HPV antigen in the peripheral blood with antigen presenting cells inthe presence of IL-7 and IL-15 and in the absence of IL-6 and/or IL-12and in the presence of co-stimulatory cells, wherein the antigenpresenting cells were previously exposed to one or more peptides,wherein the peptides comprise sequence that corresponds to at least 8contiguous amino acids of the sequence of one or both of E6 and E7 ofHPV; and (2) administering the generated or expanded population of Tcells to a subject with HPV-positive cancer.
 14. The method of claim 13,wherein the antigen presenting cells are activated T cells, dendriticcells (DC), B-blasts (BB), or peripheral blood mononuclear cells(PBMCs).
 15. The method of claim 13, wherein the co-stimulatory cellsare CD80+, CD86+, CD83+, 4-1BBL+, CD40+ cells, OX40+ cells, or acombination thereof.